Process for preparing porous collagen matrix from connective tissue

ABSTRACT

The subject invention provides a process for preparing a porous collagen matrix from connective tissue, which comprises treating connective tissue with a solution of hydrogen peroxide and/or an acidic solution so as to obtain a porous collagen matrix.

FIELD OF THE INVENTION

[0001] The subject invention relates to a process for preparing a porouscollagen matrix from connective tissue and a porous collagen matrixprepared by said process.

BACKGROUND OF THE INVENTION

[0002] Collagen is a biodegradable protein and exists in a form offibers in connective tissue of most animals. The primary function ofcollagen is to maintain the integrity of tissues and to provide tensilestrength essential to tissues. Collagen molecule is a biologicalmacromolecule composed of three polypeptide chains that twist around oneanother. Each polypeptide is composed of more than one thousand aminoacids, wherein the primary amino acids are glycine, proline andhydroxyproline. At present, more than 21 different types of collagenhave been discovered.

[0003] For applications, collagen can be manufactured in differentforms, such as sponge, gel, tube, sheet, etc. They can be applied ashemostats, wound dressings, drug carriers, scaffolds of artificialorgans, fillers to recover tissues, microcarriers and macrocarriers forsupporting cell growth, etc. In order to make the above referredcollagen matrix existing a porous structure to facilitate cellmigration, cell growth or encapsulation and release of drugs, collagenis usually isolated from connective tissue and fabricated into a porousmatrix through a lyophilization step. Generally, the matrix is treatedwith a cross-linking agent and is frozen at various temperaturesfollowed by vacuum drying during the lyophilization step.

[0004] The preparation of porous collagen matrix has been disclosed inmany prior art patents. For instance, in U.S. Pat. No. 4,193,813,comminuted collagen at pH 3.5 to pH 6.5 is crosslinked withglutaraldehyde followed by freezing at 0 to −20° C. After thawing, thewater of the frozen material is eliminated to form a sponge matrix. Thepore size of the matrix formed by this process is about 80˜1400 μm.

[0005] U.S. Pat. No. 4,412,947 relates to a process that pure insolubleparticulate collagen is suspended in a weak aqueous organic acidsolution followed by freezing at −60 to −70° C. with a temperaturereduction rate of −0.3 to −0.4° C. per minute, and then lyophilized toform a porous collagen sheet. U.S. Pat. No. 4,522,753 relates to aprocess of mixing collagen and chrondroitin sulfate to form a copolymermaterial. The material is then cross-linked by glutaraldehyde andlyophilized to form a porous matrix with a pore size of 20˜180 μm. Suchmatrix can be used as a basic material of synthetic skin grafts.

[0006] U.S. Pat. No. 4,970,298 discloses a collagen matrix prepared bydispersing collagen in an acidic solution or by mixing the collagendispersion with hyaluronic acid and fibronectin. The dispersion isfrozen at the different temperatures and then lyophilized to form aporous sponge. The sponge is cross-linked with a carbodiimide or by adehydrothermal process. The freezing temperature is −30° C. to −50° C.The pore size of the matrix obtained is about 20˜250 μm. The collagenmatrix containing hyaluronic acid or fibronectin exhibits a pore size of100˜150 μm.

[0007] U.S. Pat. No. 4,948,540 describes a process that involvesfreeze-drying the mixture of native collagen and soluble collagen fibersand compressing at a pressure of 15,000˜30,000 p.s.i. The material isthen cross-linked by dehydrothermal method to obtain a final productwhich is a sheet material with high absorptivity.

[0008] U.S. Pat. No. 5,116,552 describes a process for preparing acrack-free sponge matrix. An acidic collagen solution is frozen at −40°C. and lyophilized into a sponge. The sponge is then incubated at 105°C. for 24 hours and then cross-linked for 24 hours with glutaraldehydeto form a matrix with a pore size of 50˜120 μm. The matrix is thenimmersed in 15% alcohol. After second lyophilization at a lowertemperature of −80° C. or −135° C., a crack-free sponge matrix isobtained.

[0009] U.S. Pat. No. 5,869,080 describes a process for preparing anabsorbable implant material. A sponge matrix is formed by adding aproper amount of alcohol to the collagen dispersion in sodium hydroxide,pre-freezing it at a low temperature (about −5° C.), adding iceparticles to the dispersion, cross-linking the dispersion withhexamethylene diisocyanate (HMDI), and followed by lyophilizing thedispersion. The matrix obtained by this process exhibits a pore size of50˜400 μm.

[0010] The processes for the preparation of the collagen matrixdisclosed in the aforementioned patents comprise complicated operationsteps which include extraction and purification of collagen. Acidic oralkaline collagens are generally used and cross-linked by adehydrothermal process or by the chemical cross-linking agents, andlyophilized to obtain porous collagen matrices. The matrix productsobtained by any of these processes exhibit poor porous homogeneity.Furthermore, because most of the chemical cross-linking agents aretoxic, the application extent of these processes is limited.

[0011] To obtain a better porous homogeneity, to prevent possible toxiceffects resulted from the chemical cross-linking agents, to save vastenergy on collagen extraction, and yet to obtain a more stable porousnetwork, the subject invention proposes an improved process formanufacturing the porous collagen matrix for collagen-related productsand applications.

SUMMARY OF THE INVENTION

[0012] The subject invention provides an improved method and productsthereof that overcome the disadvantages of the conventional techniquefor preparing a collagen matrix. The operation steps of the process ofthe subject invention are simple and do not need to use a cross-linkingagent. Meanwhile, the subject invention provides a collagen matrixproduct with excellent qualities with unexpected results.

[0013] An object of the subject invention is to provide a process forpreparing a porous collagen matrix from connective tissue.

[0014] Another object of the invention is to provide a porous collagenmatrix prepared by the process of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Additional objects and features of the present invention willbecome more apparent and the invention itself will be best understoodfrom the following Detailed Description of the Invention, when read withreference to the accompanying drawings.

[0016]FIG. 1 shows the cross section of the porous collagen matrixprepared by one embodiment of the present invention illustrated in theprocess of Example 1.

[0017]FIG. 2 shows the cross section of the porous collagen matrixprepared by another embodiment of the present invention illustrated inthe process of Example 2.

[0018]FIG. 3 shows the cross section of the porous collagen matrixprepared by still another embodiment of the present inventionillustrated in the process of Example 3.

[0019]FIG. 4 shows the cross section of the porous collagen matrixprepared by one further embodiment of the present invention illustratedin the process of Example 4.

[0020]FIG. 5 shows the cross section of the porous collagen matrixprepared by one alternate embodiment of the present inventionillustrated in the process of Example 5.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The preferred embodiments of the present invention describedbelow relate particularly to a porous collagen matrix and innovativeprocesses for making same. While the description sets forth variousembodiment specific details, it will be appreciated that the descriptionis illustrative only and should not to be construed in any way aslimiting the invention. Furthermore, various applications of theinvention, and modifications thereto, which may occur to those who areskilled in the art, are also encompassed by the general conceptsdescribed below.

[0022] The process of the subject invention overcomes the disadvantagesof the conventional technique of preparing collagen matrices. Theprocess of the subject invention has the following features andadvantages: (i) the cost of manufacturing the porous collagen matrix issubstantially reduced; (ii) the preparation time is greatly decreased;(iii) the structure of the matrix is more stable than the conventionallyprepared collagen matrix; (iv) without the use of cross-linking agents;and (v) the collagen matrix prepared by the process of the subjectinvention can be readily used directly as biomedical materials orscaffolds for tissue engineering.

[0023] The subject invention utilizes animal connective tissue asstarting materials. Then the material is subject to the necessarytreatment procedures (such as the treatment with a chemical solutionand/or bactericide, and low-temperature freezing and lyophilizationunder vacuum) to form porous structure.

[0024] Therefore, it is some aspects of the subject invention to providea process for the preparation of a porous collagen matrix. Said processcomprises treating connective tissue with a solution of hydrogenperoxide so as to obtain a porous collagen matrix.

[0025] It is another aspect of the subject invention to provide aprocess for preparing a porous collagen matrix from connective tissue,wherein the process comprises treating connective tissue with an acidicsolution so as to obtain a porous collagen matrix.

[0026] It is still another aspect of the subject invention to provide aprocess for preparing a porous collagen matrix from connective tissue,wherein the process comprises treating connective tissue with an acidicsolution followed by a hydrogen peroxide solution, freezing saidconnective tissue at a proper temperature under a proper temperaturereduction rate, and lyophilizing said connective tissue so as to obtaina porous collagen matrix.

[0027] Collagen is rich in animal connective tissue. Therefore, thesubject invention may directly utilize connective tissue as the startingmaterials. The source of connective tissue may derive from animals whichhave connective tissue, such as cattle, pigs, horses, sheep, chickens,ducks, turkeys, gooses, whales, sharks, and the like. The connectivetissue suitable for the process of the subject invention includes skin,dermis, subcutaneous tissue, ligament, tendon, aponeurose, cartilage,bone tissue, and the like. The following description of dermis treatmentis an example of the subject invention. Lipid of fresh animal skin isremoved, and then the animal skin is washed a few times with saline. Thesurface layer of the animal skin is removed with a dermatome, and thenthe dermis with proper thickness is obtained. The dermis is washed withphosphate buffered saline. Solution retained on the surface of thedermis is generally removed by absorption. The dermis can be subject tothe subsequent treatments with chemical reagents.

[0028] In one embodiment, the process of the subject invention utilizesan acid to treat connective tissue so that the connective tissue can besoftened. The acids suitable for the subject invention include organicor inorganic acids. Inorganic acids may include, but are not limited to,hydrochloric acid, phosphoric acid, boric acid or sulfuric acid.

[0029] Organic acids may include, but is not limited to peracetic acid,formic acid, acetic acid, propanoic acid, butyric acid, pantanoic acid(valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthicacid), capric acid, oxalic acid, malonic acid, succinic acid, glutaricacid, adipic acid, benzoic acid, and the analogs, or derivatives of theaforementioned acids, such as methylpantanoic acid, 2-hydroxypropanoicacid (lactic acid), chlorohexanoic acid, 4-hydroxy-6-methylheptanoicacid, 2-aminopropanoic acid, 2,3-dihydroxysuccinic acid, butenedioicacid (fumaric acid or maleic acid), methylbenzoic acid, chlorobenzoicacid, hydroxybenzoic acid, phthalic acid, cyclohexane carboxylic acid,methylcyclobutane carboxylic acid, methylcyclohexane carboxylic acid,cyclopentane dicarboxylic acid, etc.

[0030] In the step of acid treatment, the proper concentration of theacid varies with the species of the acid. For instance, theconcentration of acetic acid is preferably at about 0.01 to 4M. In thatcase, dermal tissue is reacted at 4 to 45 for 1 to 150 hours. Theconcentration of hydrochloric acid is preferably at about 0.01 to 2N. Inthat case, dermal tissue is reacted at 0 to 60 for 0.1 to 150 hours.

[0031] The acid treatment can be carried out in two steps. For instance,dermal tissue is initially treated with an acidic solution at higherconcentration of 0.1 to 6M for 0.1 to 100 hours in the first step, andthen with an acidic solution at lower concentration of 0.01 to 2M for 1to 200 hours in the second step. For instance, one of the preferredembodiment of the subject invention is that connective tissue is treatedwith 1.0M of acetic acid solution on a rotator at 37 for 12 hours; thenthe treated connective tissue is placed in 0.2M of acetic acid solutionfor 72 hours so as to achieve the effects of penetration and immersionof acids in both of inner and outer parts of the connective tissue.

[0032] In the step of the acid treatment, salts can be added to theacidic solution so as to stabilize collagen molecules to create a betterporous structure in the matrix. For instance, the concentration of asalt is preferably at about 0.01 to 4M. Salts suitable for the subjectinvention include organic or inorganic salts. Inorganic salts mayinclude, but are not limited to, halide salt of alkaline or alkalineearth group, such as sodium chloride or calcium chloride. Organic saltsmay include, but are not limited to, carboxylates, such as calciumpropanoate, sodium benzoate, sodium acetate or sodium carbonate. When0.1 to 2M of sodium chloride is added to the acidic solution, the besteffect for stabilizing the matrix can be achieved and the best porositycan be obtained. For instance, in the process of the subject inventionmentioned above, a collagen matrix with stable and homogeneous pores canbe formed if 0.5M of sodium chloride is added to the lower concentration(0.2M) of acetic acid solution.

[0033] The hydrogen peroxide treatment can simultaneously kill bacteriaand produce pores in the matrix. The concentration of hydrogen peroxidesolution suitable for the process of the subject invention is 0.1% to10%, preferably 0.5% to 3.0%. This treatment can be carried out at 0 to70, preferably 4 to 60 , for 0.1 to 240 hours. Said treatment can becarried with peracetic acid, NaClO₄, periodic acid, perbromic acid,hydroxide or halide containing tertiary amino group, such asd-tubocurarine chloride, choline chloride, muscarine hydroxide,acetylcholine hydroxide, betaine, decamethonium chloride, hexamethoniumchloride, etc. In addition, other chemical agents, such as hydriodicacid, trichloromelamine, difluoroacetic acid, chlorine dioxide,polyvinyl pyrrolidone-iodine (povidone-iodine), p-aminosalicylic acid,isonicotinic acid hydrazine, sulfonamides, trimethoprim, metronidazole,4-quinolone derivatives, imidazole derivatives, azidothymidine, etc.,can be used in this step to achieve the effect similar to the hydrogenperoxide treatment.

[0034] To facilitate that the porous collagen matrix can be directlyutilized as artificial skins or tissue scaffolds, the process of thesubject invention may further comprise a step of removingnon-collagenous impurities. Said step utilizes a solution containing adetergent, such as sodium dodecyl sulphate (SDS), Tego compounds (suchas Tween 80, Triton W. R. 1339, p-isooctylpolyoxy-ethylene phenolpolymer; Triton A20), cetylpyridinium chloride, cetyltrimethyl-ammoniumbromide, dioctyl sodium sulphosuccinate, Emasol 4130 (polyoxyethylenesorbitan monoleate), Lubrol W, Nonidet P40, etc. Preferably, a solutioncontaining 0.01 to 10% of SDS can be used to treat the connective tissueat 4 to 45 for 1 to 150 hours.

[0035] The solution containing a detergent used in the process of thesubject invention may further comprise a chelating agent, such as EDTA(ethylene diamine tetra-acetic acid), DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonicacid)), CDTA (trans-1,2-diaminocyclohexantetra-acetic acid, Tiron(4,5-dihydroxybenzene-1,3-disulphonic acid, thiourea,8-hydroxyquinoline-5-sulphonic acid,3,6-disulpho-1,8-dihydroxynaphthalene, Eriochromeschwarz T(1-(1-hydroxy-2-naphthylazo)-2-hydroxy-5-nitro-4-naphthalene sulphonicacid), ammonium purpurate, etc. For instance, the chelating agent ispreferably EDTA with a concentration of 0.01 to 100 mM. Regarding theaforementioned solutions, when the connective tissue is treated with asolution containing only SDS, a collagen matrix with smaller pore sizeis obtained.

[0036] After the treatments with the aforementioned chemical agents, theconnective tissue can be subject to lyophilization. In the priorfreezing step of lyophilization, the temperature reduction rate and thefinal freezing temperature are associated with the pore size andhomogeneity of the matrix. For example, when the collagen matrix isfrozen at −20, the homogeneity of pores in the said matrix is moreproper for cell ingrowth; when the collagen matrix is frozen at 80 , thepore size of the matrix is smaller.

[0037] It is one object of the subject invention to provide the porouscollagen matrices prepared by the aforementioned processes. The porouscollagen matrix prepared by the processes of the subject invention canbe used in the preparation of artificial organs or biomedical materials,as the media for cell culture, or used in tissue engineering.

[0038] The following examples are for further illustration of theinvention but not intended to limit the invention. Any modifications andapplications by persons skilled in the art in accordance with theteachings of the invention should be within the scope of this invention.

EXAMPLE

[0039] Example 1

[0040] Lipid and fat of pigskin were removed and washed twice. Epidermisof pigskin was removed with a dermatome, and dermal tissue withthickness of 0.2 mm was selected. The dermal tissue was washed with aphosphate buffered saline solution containing 0.02% NaN₃. Solutionretained on the surface of the dermal tissue was removed by absorption.

[0041] The dermal tissue was then treated with a solution of 3% hydrogenperoxide on a rotator at 37 for 24 hours, and then washed with aphosphate buffered saline solution. A porous collagen matrix wasobtained and the cross section of the matrix was evaluated andphotographed under a light microscope as shown in FIG. 1.

Example 2

[0042] The pig dermal tissue was pre-treated according to the processdescribed in Example 1, and then treated with a solution of 3% hydrogenperoxide. The dermal tissue was then washed with an aseptic phosphatebuffered saline solution and frozen at −20 for lyophilization. A porouscollagen matrix was obtained and the cross section of the matrix wasevaluated and photographed under a light microscope as shown in FIG. 2.

Example 3

[0043] Lipid and fat of pigskin were removed and washed twice. Epidermisof pigskin was removed with a dermatome, and dermal tissue withthickness of 0.2 mm was selected. The dermal tissue was washed with aphosphate buffered saline solution containing 0.02% NaN₃. Solutionretained on the surface of the dermal tissue was removed by absorption.

[0044] The dermal tissue was then treated with a solution of 0.5Nhydrochloric acid on a rotator at 37 for 3 hours. The treated dermaltissue was then washed with an aseptic phosphate buffered salinesolution so as to remove residual chemical agents. A porous collagenmatrix was obtained and the cross section of the matrix was evaluatedand photographed under a light microscope as shown in FIG. 3.

Example 4

[0045] Lipid and fat of pigskin were removed and washed twice. Epidermisof pigskin was removed with a dermatome, and dermal tissue withthickness of 0.5 mm was selected. The dermal tissue was washed with aphosphate buffered saline solution containing 0.02% NaN₃. Solutionretained on the surface of the dermal tissue was removed by absorption.

[0046] The dermal tissue was then treatment with a solution of 0.5Nhydrochloric acid on a rotator at 37 for 6 hours. The dermal tissue wasthen washed with an aseptic phosphate buffered saline solution to removeresidual chemical agents. Solution retained on the surface of the dermaltissue was removed by absorption. The dermal layer was then frozen at−20 for lyophilization. A porous collagen matrix was obtained and thecross section of the matrix was evaluated and photographed under a lightmicroscope as shown in FIG. 4.

Example 5

[0047] Lipid and fat of pigskin were removed and washed twice. Epidermisof pigskin was removed with a dermatome, and dermal tissue withthickness of 0.5 mm was selected. The dermal tissue was washed with aphosphate buffered saline solution containing 0.02% NaN₃. Solutionretained on the surface of the dermal tissue was removed by absorption.

[0048] The dermal tissue was then treated with a solution of 1.0M aceticacid on a rotator at 37 for 12 hours. The dermal tissue was thentransferred to a solution of 0.2M acetic acid containing 0.5M sodiumchloride and reacted for 72 hours. After treatment with acetic acidsolution, the dermal tissue was transferred to a solution of 1% hydrogenperoxide for reaction for 24 hours and then to a solution containing SDSand EDTA for reaction at 37 for 24 hours.

[0049] After completing the aforementioned treatments with the chemicalsolutions, the dermal tissue was washed with aseptic phosphate bufferedsaline solution to remove the residual chemical agents. The dermaltissue was then frozen at −20 for lyophilization. A porous collagenmatrix was obtained and the cross section of the matrix was evaluatedand photographed under a light microscope. As shown in FIG. 5, thehomogeneity of pores and the pore size of the matrix are very suitablefor applications.

[0050] Although preferred embodiments of the invention have beendescribed in detail, including a porous collagen matrix and methods ofmanufacturing thereof, certain variations and modifications will beapparent to those skilled in the art, including embodiments that do notprovide all of the features and benefits described herein. Accordingly,the scope of the present invention is not to be limited by theillustrations or the foregoing descriptions thereof, but rather solelyby reference to the appended claims.

We claim:
 1. A process for preparing a porous collagen matrix fromconnective tissue, said process comprising treating connective tissuewith a solution of hydrogen peroxide effective for preparing the porouscollagen matrix.
 2. The process according to claim 1, wherein thesolution of hydrogen peroxide has a concentration between 0.1 and 10%.3. The process according to claim 1, wherein the connective tissue istreated at a temperature ranging from 4 to
 60. 4. The process accordingto claim 1, the process further comprising steps of freezing theconnective tissue treated with a solution of hydrogen peroxide, whereinthe step of freezing is carried out at a proper temperature with aproper temperature reduction rate, and lyophilizing the connectivetissue effective for preparing the porous collagen matrix.
 5. A processfor preparing a porous collagen matrix from connective tissue, saidprocess comprising treating connective tissue with an acidic solutioneffective for preparing the porous collagen matrix.
 6. The processaccording to claim 5, wherein the acidic solution is selected from agroup consisting of peracetic acid, hydrochloric acid, oxalic acid,acetic acid and sulfuric acid.
 7. The process according to claim 5,wherein the acidic solution has a concentration of 0.01 to 4M.
 8. Theprocess according to claim 5, wherein the acidic solution comprises atleast a salt with a proper concentration.
 9. The process according toclaim 8, wherein the salt has a proper concentration of 0.01 to 4M ofsodium chloride.
 10. The process according to claim 5, the processfurther comprising steps of freezing the connective tissue treated withan acidic solution, wherein the step of freezing is carried out at aproper temperature with a proper temperature reduction rate, andlyophilizing the connective tissue effective for preparing the porouscollagen matrix.
 11. A process for preparing a porous collagen matrixfrom connective tissue, the process comprising steps of treatingconnective tissue with an acidic solution followed by a solution ofhydrogen peroxide, freezing said connective tissue at a propertemperature under a proper temperature reduction rate, and lyophilizingsaid connective tissue effective for preparing the porous collagenmatrix.
 12. The process according to claim 11, wherein the solution ofhydrogen peroxide has a concentration between 0.1 and 10%.
 13. Theprocess according to claim 11, wherein the connective tissue is treatedat a temperature ranging from 4 to
 60. 14. The process according toclaim 11, wherein the acidic solution is selected from a groupconsisting of peracetic acid, hydrochloric acid, oxalic acid, aceticacid and sulfuric acid.
 15. The process according to claim 11, whereinthe treatment with an acidic solution is divided into two steps, a firststep comprising treating the connective tissue with an acetic acidsolution of 0.1 to 6M and a second step comprising treating theconnective tissue with an acetic acid solution of 0.01 to 2M.
 16. Theprocess according to claim 15, wherein the acetic acid solution of 0.01to 2M comprises at least a salt.
 17. The process according to claim 16,wherein the salt comprises sodium chloride of 0.01 to 4M.
 18. Theprocess according to claim 11, the process further comprising a step oftreating the connective tissue with a solution containing SDS (sodiumdodecyl sulphate).
 19. The process according to claim 18, wherein thesolution contains 0.01 to 10% of SDS.
 20. The process according to claim19, wherein the solution contains a chelating agent.
 21. The processaccording to claim 20, wherein the chelating agent is EDTA (ethylenediamine tetra-acetic acid) with a concentration of 0.01 to 100 mM.
 22. Aporous collagen matrix prepared by the process according to claim
 1. 23.A porous collagen matrix prepared by the process according to claim 4.24. A porous collagen matrix prepared by the process according to claim5.
 25. A porous collagen matrix prepared by the process according toclaim
 7. 26. A porous collagen matrix prepared by the process accordingto claim
 9. 27. A porous collagen matrix prepared by the processaccording to claim
 10. 28. A porous collagen matrix prepared by theprocess according to claim
 11. 29. A porous collagen matrix prepared bythe process according to claim
 15. 30. A porous collagen matrix preparedby the process according to claim
 17. 31. A porous collagen matrixprepared by the process according to claim
 20. 32. A porous collagenmatrix prepared by the process according to claim 22.